Exocrine pancreatic acinar cells manufacture, store and release large quantities of hydrolytic enzymes into the intestine that are essential for nutrient digestion and absorption. As food is derived from cellular material, acinar cells retain protective mechanisms to ensure digestive enzymes are not prematurely activated until reaching the intestine. Indeed, aberrant dysregulation of the acinar secretory pathway and premature activation of enzymes has been tied to the development of pancreatitis and pancreatic cancer, which affect over 48,000 Americans each year. It is widely believed that premature digestive enzyme activation is caused by abnormal interactions of the secretory and lysosomal pathways in acini. Thus, knowledge of the basic molecular mechanisms which orchestrate the normal interactions of these pathways is critical to understanding the pathophysiology of pancreatic disease. This proposal investigates a previously unrecognized and important regulatory pathway by which acinar cells modulate digestive enzyme trafficking within the secretory and lysosomal pathways and accordingly regulate digestive enzyme secretion. We recently reported that acinar cells express two distinct populations of zymogen granules (ZGs) based on the expression of the exocytic regulatory proteins VAMP2 and 8. Moreover, our data indicate that maturation of VAMP8 ZGs is directed by Tumor Protein D52 (aka CRHSP-28), which we have shown is uniquely expressed in acini and directly regulates Ca2+-dependent secretion. Unexpectedly, D52 was localized to a unique endosome and lysosome related compartment in acini that we term the endo-lysosomal compartment. Moreover, in CHO-K1 cells, D52 directly regulates lysosmal membrane exocytosis and when over-expressed induces a massive accumulation of cytoplasmic vesicles. This proposal aims to delineate the precise functional roles of VAMP2- and VAMP8- positive ZGs in the acinar secretory response and will test the central hypothesis that D52-regulated trafficking through the endo-lysosomal system controls the biogenesis/maturation of VAMP8-positive ZGs within a unique lysosome-related pathway. Specific Aim 1 will test the hypothesis that VAMP2-positive ZGs mediate the early immediate release of digestive enzymes, whereas VAMP8-positive ZGs mediate the sustained plateau phase of zymogen secretion following acinar stimulation. Specific Aim 2 will test the hypothesis that activation of PIKfyve to produce PtdIns(3,5)P2 in acini regulates endo-lysosomal trafficking to the lysosome and inhibits apical secretion during acinar hyperstimulation. Specific Aim 3 will test the hypothesis that D52 activation of the minor regulated pathway mediates the rapid insertion of important regulatory proteins into the apical membrane necessary for granule exocytosis. Understanding the basic molecular principles of how acini uniquely orchestrate the secretory or lysosomal compartments is key to the development of therapeutic strategies aimed at treating pancreatic disease. PUBLIC HEALTH RELEVANCE: Pancreatitis and pancreatic cancer are the two major diseases of the exocrine pancreas that afflict over 48,000 Americans each year with high mortality rates. The experiments outlined in this proposal will provide a basic understanding of a key regulatory mechanism by which acinar cells of the pancreas regulate the manufacture and secretion of digestive enzymes as well as the aberrant changes in these processes leading to pancreatic disease. These studies are essential to identify important cellular targets that may be therapeutically manipulated to treat pancreatic disease.